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All four putative ligand-binding domains in megalin contain pathogenic epitopes capable of inducing passive Heymann
H Yamazaki1, R Ullrich, M Exner
1Department of Pathology, University of California San Diego, La Jolla 92093-0651, USA.
Abstract:
Megalin (gp330) is the main target antigen involved in the induction of Heymann nephritis (HN), a rat model of human membranous nephropathy. Its large extracellular region contains four putative ligand-binding domains separated by spacer regions. Previously, it was reported that the second ligand-binding domain (LBD II) of megalin is involved in the pathogenesis of passive HN because it is capable of binding antibodies in vivo and initiating formation of immune deposits (ID). This study explores the possibility that pathogenic epitopes might also be present in the other putative ligand-binding domains. Recombinant fragments of ligand-binding domains (LBD) I through IV expressed in a baculovirus system were used to generate polyclonal domain-specific antibodies. Antibodies raised against each of the recombinant megalin fragments reacted preferentially with its respective antigen and with whole megalin by immunoblotting. Each of the antibodies also gave a characteristic brush-border staining for megalin by indirect immunofluorescence on rat kidney. When rats were injected with the domain-specific antibodies to test their ability to produce passive HN, glomerular ID were present in kidneys of all injected animals. The staining pattern in glomeruli of rats injected with LBD I, III, or IV was similar to that obtained with antibodies to LBD II. It is concluded that passive HN can be induced with antibodies against LBD I, III, and IV, as well as LBD II, and that each of the ligand-binding domains contains a pathogenic epitope. These findings provide further evidence for the multiple epitope model of HN.
Insights
Heymann nephritis (HN) involves megalin (gp330). Antibodies targeting any of megalin's four ligand-binding domains (LBD I-IV) can induce passive HN, indicating multiple pathogenic epitopes.
Area of Science:
- Nephrology
- Immunology
- Molecular Biology
Background:
- Megalin (gp330) is the primary target antigen in Heymann nephritis (HN), a rat model for human membranous nephropathy.
- The second ligand-binding domain (LBD II) of megalin was previously identified as crucial for passive HN pathogenesis due to antibody binding and immune deposit formation.
- The presence of pathogenic epitopes in other ligand-binding domains of megalin remained unexplored.
Purpose of the Study:
- To investigate whether pathogenic epitopes are present in ligand-binding domains (LBD) I, III, and IV of megalin, in addition to LBD II.
- To determine if antibodies against LBD I, III, and IV can induce passive Heymann nephritis.
Main Methods:
- Recombinant fragments of megalin's ligand-binding domains (LBD I-IV) were expressed using a baculovirus system.
- Polyclonal domain-specific antibodies were generated against these recombinant fragments.
- Passive Heymann nephritis was induced in rats by injecting these domain-specific antibodies, and kidney tissues were analyzed for immune deposits and staining patterns.
Main Results:
- Antibodies raised against each LBD fragment showed specific reactivity with their respective antigens and whole megalin.
- Immunofluorescence confirmed characteristic brush-border staining for megalin in rat kidneys using each domain-specific antibody.
- Injection of antibodies against LBD I, III, and IV successfully induced passive Heymann nephritis, with immune deposits observed in glomeruli, similar to antibodies against LBD II.
Conclusions:
- Passive Heymann nephritis can be induced by antibodies targeting not only LBD II but also LBD I, III, and IV of megalin.
- Each of the four ligand-binding domains of megalin contains at least one pathogenic epitope.
- These findings support a multiple epitope model for the induction of Heymann nephritis.